Normalization strategies differently affect circulating miRNA profile associated with the training status

Abstract

MicroRNAs are fine regulators of the whole-body adaptive response but their use as biomarkers is limited by the lack of standardized pre- and post-analytical procedures. This work aimed to compare different normalization approaches for RT-qPCR data analyses, in order to identify the most reliable and reproducible method to analyze circulating miRNA expression profiles in sedentary and highly-trained subjects. As the physically active status is known to affect miRNA expression, they could be effective biomarkers of the homeostatic response. Following RNA extraction from plasma, a panel of 179 miRNAs was assayed by RT-qPCR and quantified by applying different normalization strategies based on endogenous miRNAs and exogenous oligonucleotides. hsa-miR-320d was found as the most appropriate reference miRNA in reducing the technical variability among the experimental replicates and, hence, in highlighting the inter-cohorts differences. Our data showed an association between the physically active status and specific skeletal muscle- and bone-associated circulating miRNAs profiles, revealing that established epigenetic modifications affect the baseline physiological status of these tissues. Since different normalization strategies led to different outputs, in order to avoid misleading interpretation of data, we remark the importance of the accurate choice of the most reliable normalization method in every experimental setting.

MicroRNAs as Appropriate Discriminators in Non-Specific Alpha-Fetoprotein (AFP) Elevation in Testicular Germ Cell Tumor Patients

“…First, the lack of a pre-operative serum samples to confirm the detection of miR-371a-3p in the patient which enables us only to be able to indirectly conclude the high specificity of this biomarker under this condition based on our previous findings [15]. Another limitation is the use of the proper housekeeping gene from serum, as some newer studies also reported issues about pre-analytical influence of haemolysis on the concentration of the serum-based microRNAs [20,21]. In addition, we cannot rule out a lack of expression of this microRNA in the cancer tissue of our patient.…”

Challenges in Using Circulating Micro-RNAs as Biomarkers for Cardiovascular Diseases

“…However, without added spike-ins and standard curves, all techniques are mostly based on relative quantification, and the levels of miRNAs are presented as a "fold change" between paired samples, and not as an absolute unit, introducing bias when results from different studies are compared. Normalization strategies usually lead to different results and therefore to avoid misleading interpretation of data it is essential to choose the most reliable normalization method in each experimental setting [130].…”

“…The endogenous reference genes that are routinely used for data normalization have been recently reviewed in [133]. However, until today a reliable "housekeeping" c-miRNA has not been identified [134], suggesting that correcting plasma/serum volume is the best method of normalization, as the volume of plasma/serum is clinically standard for other biomarkers [130]. Moreover, in a disease state including CVDs, higher amounts of miRNAs are released in the circulation compared to healthy individuals and thus equal volumes of starting material should be used instead of using the same amount of total RNA [79].…”

Circulating Brain‐Enriched MicroRNAs for Detection and Discrimination of Idiopathic and Genetic Parkinson's Disease

“…Stable reference (miR‐103a‐3p, miR‐191‐5p, miR‐425‐5p, miR‐223‐3p, miR‐423‐3p; Exiqon's biofluid guidelines and refs. and hemolysis (miR‐23a, miR‐451a) miRNA controls were included in the plasma analysis. The relative expression level of miRNAs was calculated using the 2 −ΔΔCt method.…”